Orientation of bound ligands in mannose-binding proteins. Implications for multivalent ligand recognition

Mannose-binding proteins (MBPs) are C-type animal lectins that recognize high mannose oligosaccharides on pathogenic cell surfaces. MBPs bind to their carbohydrate ligands by forming a series of Ca(2+) coordination and hydrogen bonds with two hydroxyl groups equivalent to the 3- and 4-OH of mannose. In this work, the determinants of the orientation of sugars bound to rat serum and liver MBPs (MBP-A and MBP-C) have been systematically investigated. The crystal structures of MBP-A soaked with monosaccharides and disaccharides and also the structure of the MBP-A trimer cross-linked by a high mannose asparaginyl oligosaccharide reveal that monosaccharides or alpha1-6-linked mannose bind to MBP-A in one orientation, whereas alpha1-2- or alpha1-3-linked mannose binds in an orientation rotated 180 degrees around a local symmetry axis relating the 3- and 4-OH groups. In contrast, a similar set of ligands all bind to MBP-C in a single orientation. The mutation of MBP-A His(189) to its MBP-C equivalent, valine, causes Man alpha 1-3Man to bind in a mixture of orientations. These data combined with modeling indicate that the residue at this position influences the orientation of bound ligands in MBP. We propose that the control of binding orientation can influence the recognition of multivalent ligands. A lateral association of trimers in the cross-linked crystals may reflect interactions within higher oligomers of MBP-A that are stabilized by multivalent ligands.     

Lectin-Carbohydrate Interactions: Fine Specificity Difference Between Two Mannose-Binding Proteins

Two types of rat mannose-binding proteins (MBPs), MBP-A (serum type) and MBP-C (liver type), have similar binding specificity for monosaccharide and similar binding site construct according to the X-ray structure, but exhibit different affinity toward natural oligosaccharides and glycoproteins. To understand the basis for this phenomenon, we used cloned fragment of MBP-A and -C (entire carbohydrate-recognition domain and a short connecting piece) that exists as stable trimers in various binding studies. Binding of a number of mannose-containing di- and tri-saccharides and high-mannose type oligosaccharides indicated that MBP-C has an extended binding area of weak interaction with the second and the third mannose residues, whereas MBP-A recognizes just a single mannose residue. In addition, MBP-C has a weak secondary binding site some 25 Å away from the primary site. These findings explain the higher affinity of MBP-C for natural high-mannose type oligosaccharides as compared to MBP-A. A huge affinity differential manifested by natural glycoproteins (e.g., inhibitory potency of thyroglobulin is ~200 fold higher for MBP-C than for MBP-A in a solid-phase assay) may be due to steric hindrance experienced by MBP-A in the competition assay, and suggests different arrangement of subunit in the MBP trimers.     

Differential recognition of core and terminal portions of oligosaccharide ligands by carbohydrate-recognition domains of two mannose-binding proteins

Two different mannose-binding proteins (MBP-A and MBP-C), which show 56% sequence identity, are present in rat serum and liver. It has previously been shown that MBP-A binds to a range of monosaccharide-bovine serum albumin conjugates, and that, among oligosaccharide ligands tested, preferential binding is to terminal nonreducing N-acetylglucosamine residues of complex type N-linked oligosaccharides. In order to compare the binding specificity of MBP-C, an expression system has been developed for production of a fragment of this protein which contains the COOH-terminal carbohydrate-recognition domain. After radioiodination, the domain has been used to probe natural glycoproteins, neoglycoproteins, and neoglycolipids. Like MBP-A, MBP-C binds several different monosaccharides conjugated to bovine serum albumin, including mannose, fucose, and N-acetylglucosamine, although binding to the last of these is relatively weaker than observed for MBP-A. The results of binding to natural glycoproteins and to neoglycolipids containing oligosaccharides derived from these proteins are most compatible with the interpretation that MBP-C interacts primarily with the trimannosyl core of complex N-linked oligosaccharides, with additional ligands being terminal fucose and perhaps also peripheral mannose residues of high mannose type oligosaccharides. This binding specificity is thus quite distinct from that of MBP-A. The presence of multiple MBPs with distinct binding specificities in preparations derived from serum and liver explains conflicting conclusions which have been reached about carbohydrate recognition by these proteins.     

Difference in binding-site architecture of the serum-type and liver-type mannose-binding proteins

The carbohydrate-recognition domains (CRDs) of the serum-type and the liver-type mannose-binding proteins (MBPs) from rat display different binding characteristics toward mannose-rich oligosaccharides derived from N-glycosides, despite the overall similarity in their binding site architecture, oligomeric status and actual binding specificity at the monosaccharide level. We found that the liver-type MBP CRD of rat (MBP-C) bound methyl glycosides of certain mannobioses and -trioses, which are part of the mannose-rich N-glycoside, more tightly than methyl α-mannopyranoside. In contrast, the serum-type MBP CRD of rat (MBP-A) bound all the methyl glycosides of manno-oligosaccharide and methyl α-mannopyranoside with similar affinities. The mannobiose and -triose most strongly bound to MBP-C CRD were Manα(1-2)Manα-OMe and Manα (1-2)Manα(1-6)Manα-OMe, respectively. From these and other data, we postulate that the binding site of MBP-C has an extended area of interaction, probably the size of a mannotriose, while MBP-A interacts essentially with one mannose residue. Abbreviations: MBP, mannose-binding protein; CRD, carbohydrate-recognition domain; BSA, bovine serum albumin; TFA-ah, 6-(trifluoroacetyl)aminohexyl; PNP, p-nitrophenyl This revised version was published online in November 2006 with corrections to the Cover Date.     

Serum lectin with known structure activates complement through the classical pathway

Serum mannan-binding protein (MBP), a lectin specific for mannose and N-acetylglucosamine, was revealed to activate the complement system as measured by passive hemolysis using sheep erythrocytes coated with yeast mannan. In contrast, rat liver MBP, which shares many properties in common with serum MBP, could not activate complement at all. The activation by serum MBP was inhibited effectively by the presence of haptenic sugars and dependent absolutely upon the presence of C4, indicating that the activation is initiated by the sugar binding activity of MBP and proceeds through the classical pathway. The 25 NH2-terminal amino acid sequence of rat serum MBP determined in this study was completely matched with that of MBP-A deduced from cDNA sequence by Drickamer et al. (Drickamer, K., Dordal, M. S., and Reynolds, L. (1986) J. Biol. Chem. 261, 6878-6887), revealing that MBP-A is in fact identical with serum MBP. On the basis of the knowledge of primary structures and physicochemical properties of rat serum and liver MBPs, a possible mechanism of the complement activation by serum MBP is discussed with reference to close similarity in the gross structures of serum MBP and C1q.     

Rhesus monkey gastroenteropancreatic hormones: relationship to human sequences

The amino acid sequences of the gastroenteropancreatic peptides of Old World mammals are generally well-conserved. However, only the glucagons and vasoactive intestinal polypeptides (VIP) have been shown to be identical among the species studied to date. Rhesus monkey (Macaca mulatta) insulin has been shown to be identical with human insulin. The question addressed in this study is whether other gastroenteropancreatic peptides are identical to the human peptides. Purification and sequencing of glucagon, pancreatic polypeptide, VIP and insulin confirmed their identity with the corresponding human peptides. However, the 17 amino acid monkey gastrin is identical to dog gastrin and differs from human gastrin by substitution of methionine for leucine at position 5 from the N-terminus and alanine for glutamic acid in position 10. If additional rhesus monkey tissues become available, it would be of interest to determine whether other gastrointestinal peptides also differ from the corresponding human peptides.     

Cloning and sequencing of rhesus monkey pepsinogen A cDNA

The complete nucleotide sequence of Rhesus monkey (Macaca mulatta) pepsinogen A (PGA) cDNA was determined from two partially overlapping cDNA clones, covering the whole coding sequence and part of the flanking sequences. The nucleotide and deduced amino acid sequences were compared to known PGA sequences from other species. The degree of similarity with human PGA appeared to be 96% at the nucleotide sequence level and 94% at the amino acid sequence level. In the coding region the divergence was highest in the activation peptide. The amino acid sequence similarity between Japanese monkey (Macaca fuscata) PGA and Rhesus monkey PGA was shown to be 99%. Using the cDNA as probe in Southern hybridization of EcoRI-digested human and Rhesus monkey genomic DNAs, PGA patterns with inter-individual differences were observed. The hybridization patterns are compatible with the existence of a PGA multigene family in both species.     

cDNAs and deduced amino acid sequences of subunits in the binding component of mouse bactericidal factor, Ra-reactive factor: similarity to mannose-binding proteins.

The complement-dependent bactericidal factor, Ra-reactive factor, binds specifically to Ra polysaccharide, which is common to some strains of Gram-negative enterobacteria, and its is a complex of proteins composed of a polysaccharide-binding component and a component that is presumably responsible for the complement activation. The former component consists of two different 28-kDa polypeptides, P28a and P28b. We determined the partial amino acid sequences of P28a and P28b, and the results indicated that these polypeptides were similar to two species of mannose-binding protein, MBP-C and MBP-A (alternative names, liver and serum mannan-binding proteins, respectively), which have been isolated from rat liver and/or serum [Drickamer, K., Dordal, M. S., & Reynolds, L. (1986) J. Biol. Chem. 261, 6878-6887; Oka, S., Itoh, N., Kawasaki, T., & Yamashina, I. (1987) J. Biochem. 101, 135-144]. Thus, we cloned the respective cDNAs, using as probes synthetic oligonucleotides for which the sequences had been deduced from the amino acid sequences of P28a and P28b and of rat MBP cDNAs. The primary structures of P28a and P28b deduced from the cloned cDNAs are homologous to one another. They have three domains, a short NH2-terminal domain, a collagen-like domain, and a domain homologous to regions of some carbohydrate-binding proteins, as has been reported for rat MBPs. Southern and Northern blotting analyses using these cDNAs indicated that the P28a and P28b polypeptides are the products of two unique mouse genes which are expressed in hepatic cells.     

Myelin basic protein is affected by reduced synthesis of myelin proteolipid protein in the jimpy mouse.

Myelin basic proteins (MBPs) from 6-day-old, 10-day-old, 20-day-old and adult normal mouse brain were compared with those from 20-day-old jimpy (dysmyelinating mutant) mouse brain to determine the effect of reduced levels of proteolipid protein (PLP) on MBPs. Alkaline-urea-gel electrophoresis showed that 6-day-old and 10-day-old normal and jimpy MBPs lacked charge microheterogeneity, since C8 (the least cationic of the components; not be confused with complement component C8) was the only charge isomer present. In contrast, MBPs from 20-day-old and adult normal mouse brain displayed extensive charge microheterogeneity, having at least eight components. A 32 kDa MBP was the major isoform observed on immunoblots of acid-soluble protein from 6-day-old and 10-day-old normal and 20-day-old jimpy mouse brain. There were eight bands present in 20-day-old and adult normal mouse brain. Purified human MBP charge heteromers C1, C2, C3 and C4 reacted strongly with rat 14 kDa MBP antiserum, whereas the reaction with human C8 was weak. This suggested that MBPs from early-myelinating and jimpy mice did not react to MBP antisera because C8 was the major charge isomer in these animals. Purification of MBPs from normal and jimpy brain by alkaline-gel electrophoresis showed that both normal and jimpy MBPs have size heterogeneity when subjected to SDS/PAGE. However, the size isoforms in normal mouse brain (32, 21, 18.5, 17 and 14 kDa) differed from those in jimpy brain (32, 21, 20, 17, 15 and 14 kDa) in both size and relative amounts. Amino acid analyses of MBPs from jimpy brain showed an increase in glutamic acid, alanine and ornithine, and a decrease in histidine, arginine and proline. The changes in glutamic acid, ornithine and arginine are characteristic of the differences observed in human C8 when compared with C1.     

Genomic DNA sequence of Rhesus (M. mulatta) cystic fibrosis (CFTR) gene

Cystic fibrosis is a common human genetic disease caused by mutations in CFTR, a gene that codes for a chloride channel that is regulated by phosphorylation and cytosolic nucleotides. As part of a program to discover natural animal models for human genetic diseases, we have determined the genomic sequence of CFTR in the Rhesus monkey, Macaca mulatta. The coding region of rhesus CFTR is 98.3% identical to human CFTR at the nucleotide level and 98.2% identical and 99.7% similar at the amino acid level. Partial sequences of flanking introns (5582 base pair positions analyzed) revealed 91.1% identity with human introns. Relative to rhesus intronic sequence, the human sequences had 27 insertions and 22 deletions. Primer sequences for amplification of rhesus genomic CFTR sequences are provided. The accession number is AF013753 (all 27 exons and some flanking intronic sequence). Received: 27 August 1992 / Accepted: 5 December 1997     

恒河猴tPA基因的克隆、测序与真核表达

目的对恒河猴tPA编码区cDNA进行测序和表达.方法采用RT-PCR方法从恒河猴淋巴细胞中扩增tPA基因,将获得的cDNA克隆于T载体,序列确定后再克隆至真核表达载体.结果测序结果表明恒河猴tPAcDNA编码区与人tPAcDNA编码区的核苷酸序列同源性为96%,由此所推导的氨基酸序列的同源性为97.5%.随后将恒河猴tPAcDNA克隆于真核表达载体,转染CHO细胞后成功表达出了有活性的tPA.培养上清检测结果显示其活性约为50?U/ml,略低于人tPA在CHO细胞中表达产物的活性.结论本研究首次报道了恒河猴tPA基因编码区的全长cDNA序列并获得了有活性的恒河猴tPA真核表达产物.将为进一步比较灵长类动物间tPA的生物学特性奠定基础.     

Sequencing and cloning of the cDNA of guinea pig eosinophil major basic protein.

Major basic protein (MBP) purified from guinea pig eosinophils elicited histamine release from rat peritoneal mast cells at concentrations higher than 3 micrograms/ml both in the presence and in the absence of extracellular Ca2+. After reverse-phase high-performance liquid chromatography, it was revealed that MBP was composed of two different proteins with quite similar molecular weights and pI values, although the amino acid compositions were slightly different. The partial amino acid sequence of one of these MBPs was determined and the primers for the polymerase chain reaction (PCR) were synthesized according to the partial amino acid sequence. Using these primers and the cDNAs obtained from guinea pig eosinophils, the PCR was carried out in order to synthesize the hybridization probe of MBP for screening the cDNA library. After screening with 8 x 10(5) clones, a positive clone, which encoded a full length of pre-proMBP, was obtained. According to the sequencing data of this clone, it was revealed that pre-proMBP was composed of 3 domains; signal peptide, acidic domain and mature MBP. The predicted pI value of mature MBP was 11.7, though that of proMBP was 7.8. The homology in the amino acid sequence between guinea pig proMBP and human proMBP was 49.4%, while guinea pig mature MBP was more homologous (58%) to human mature MBP.     

Sequence and expression of human protein kinase C-epsilon.

Two human homologues of protein kinase C-epsilon (E1 and E2) were isolated from two distinct cDNA libraries. Sequence comparisons to PKC-epsilon cDNAs from several species indicated that each of these human epsilon clones contained cloning artifacts. Thus, a composite PKC-epsilon (E3) clone was derived from clones E1 and E2. Human PKC-epsilon (E3) has an overall sequence identity of 90-92% at the nucleotide level compared to the previously characterized mouse, rat and rabbit clones. At the amino acid level, the deduced human epsilon sequence shows a 98-99% identity with the mouse, rat and rabbit sequences. Expression of the human PKC-epsilon clone in Sf9 cells confirmed that the recombinant protein displayed protein kinase C activity and phorbol ester binding activity. The recombinant protein was also recognized by two distinct epsilon-specific polyclonal antibodies.     

Comparison of the amino acid and nucleotide sequences between human and two guinea pig major basic proteins

By means of reverse-phase HPLC, 2 different proteins were obtained from apparently purified pig eosinophil major basic protein (MBP) and these proteins were named GMPB1 and GMBP2. It was revealed that these 2 components of MBP have similar molecular weights and pI values, although the amino acid compositions were slightly different. In the previous study, we cloned and sequenced GMPB1 cDNA. Here we obtained another clone by plaque hybridization using a screening probe synthesized by means of polymerase chain reaction. After sequencing, it became apparent that this clone corresponded to GMBP2. As in the case of GMBP1, the cDNA of GMBP2 encoded pre-proGMBP2 with 3 domains; signal peptide, acidic pro-portion, and mature GMBP2. By comparing the sequences of GMBP1 and GMBP2, it was revealed that the proteins were quite similar to each other. In addition, their sequences also resembled those of human MBP, especially in the basic domain of mature protein; but no such similarity existed in the pro-portion. Although the molecular weights determined by SDS-PAGE of guinea pig and human MBPs were 11,000 and 9,300, respectively, the calculated molecular weights of these 3 MBPs were all 13.8 kDa. The calculated pI values of GMBP1, GMBP2 and human MBP were 11.7, 11.3 and 11.6, respectively. By means of Harr plot analysis, it was revealed that the amino acid sequences, not only in signal peptides but also in the basic domains of mature proteins, were well conserved between guinea pig and human MBPs.     

Molecular cloning of three nonhuman primate follicle stimulating hormone beta-subunit cDNAs

The follicle stimulating hormone (FSH) beta-subunit cDNAs were cloned and sequenced for an old world primate, the rhesus monkey (Macaca mulatta), and two New World primates, the common marmoset (Callithrix jacchus) and pygmy marmoset (Cebuella pygmaea). The cDNA and predicted amino acid sequences of the rhesus monkey FSH beta-subunit were related most closely to the human FSH beta-subunit (> 96% identity). The common and pygmy marmosets have identical FSH beta-subunit cDNAs, whereas the marmoset FSH beta-subunit diverges from the rhesus and human molecules with less than 93% identity. These results have significance for the implementation of assisted reproductive technologies in the nonhuman primate as well as the evolution of genes encoding reproductive hormones.     

Characterization of Basic Proteins from Goldfish Myelin

Abstract: Myelin basic protein (MBP) from common goldfish ( Carassius auratus ) myelin was extracted with dilute mineral acid. Immunological cross-reactivity of the goldfish MBP, with polyclonal antisera raised against bovine MBP, suggested that the goldfish protein has epitopes for these antibodies. It also reacted with a monoclonal antibody specific for a seven amino acid epitope (130–137) conserved in the MBP of most mammalian species. To characterize the charge heterogeneity of this protein, we iodinated the protein with ¹²⁵I and chromatographed it on a carboxymethyl cellulose-52 column together with a nonlabeled acid soluble fraction prepared from human white matter as a carrier protein. All of the goldfish protein was recovered in the unbound fraction, demonstrating that it was less cationic than the carrier protein (human MBP). We have also examined the urea alkaline gel profile of the goldfish MBP together with the human C-1, C-2, C-3, C-4, and C-8 components. The results from these experiments indicated that this MBP extracted from goldfish brain myelin lacked the microhet-erogeneity that is associated with MBPs from higher vertebrates. The MBPs from goldfish myelin were separated into their isoforms by reversed-phase HPLC. Amino acid compositions were determined for both the 17- and 14-kDa goldfish proteins. Amino acid analysis revealed similarities with the compositions of other MBPs; however, the serine content in both the 17- and 14-kDa proteins was higher than that of the human C-1, the mouse C-1 protein, and the shark proteins. The HPLC-purified 14-kDa goldfish protein was chemically cleaved with CNBr for partial sequence analysis. Even from the limited sequence obtained, the sequence ATAST was found in goldfish, which is also present in human, rabbit, and guinea pig MBPs.     

Characterization of murine mannose-binding protein genes Mbl1 and Mbl2 reveals features common to other collectin genes

Mannose-binding protein (MBP) is a member of a family of collagenous lectins (collectins), which are believed to play an important role in first-line host defense. In this study, the two genes encoding MBP in mice-Mbl1 and Mbl2-have been isolated and their exon-intron structure studied to understand their evolutionary relationship to the single human (MBL) and the two rat MBP genes. Mouse Mbl1 and Mbl2 have five and six exons, respectively. The structure of the mouse Mbl genes is similar to that of the rat and human MBP genes and shows homology to the other collectin genes, with the entire carbohydrate recognition domain being encoded in a single exon and all introns being in phase 1. The MBP encoded by mouse Mbl1 with three cysteines in the first coding exon, like the rat Mbl1 and human MBL, is capable of a higher degree of multimerization and has apparent ability to fix complement in the absence of antibody or C1q. However, the structural features of other exons, that is, the larger size of collagen domain region in the first coding exon (64 bp in Mbl2 vs 46 bp in Mbl1) and the smaller size of the exon encoding the trimerization domain (69 bp in Mbl2 vs 75 bp in Mbl1) reveal that the single human MBL gene is closely related to rodent Mbl2 rather than rodent Mbl1. The findings in this study suggest that in contrast to the evolution of another collectin gene-bovine surfactant protein-D-which duplicated in bovidae after divergence from humans, MBP gene most likely duplicated prior to human-roden divergence, and that the human homolog to Mbl1 was perhaps lost during evolution.The nucleotide sequence data reported in this paper have been submitted to Genbank and have been assigned the accession numbers U09006-U09017.